Pilar J. Oviedo

Estradiol selectively stimulates endothelial prostacyclin production through estrogen receptor-α

Estradiol (E(2)) acts on the endothelium to promote vasodilatation through the release of several compounds, including prostanoids, which are products of arachidonic acid metabolism. Among these, prostacyclin (PGI2) and thromboxane A2 (TXA2) exert opposite effects on vascular tone. The role of different estrogen receptors (ERs) in the PGI2/TXA2 balance, however, has not been fully elucidated. Our study sought to uncover whether E(2) enhances basal production of PGI2 or TXA2 in cultured human umbilical vein endothelial cells (HUVECs), to analyze the enzymatic mechanisms involved, and to evaluate the different roles of both types of ERs (ERalpha and ERbeta). HUVECs were exposed to E(2), selective ERalpha (1,3,5-tris(4-hydroxyphenyl)-4-propyl-1h-pyrazole, PPT) or ERbeta (diarylpropionitrile, DPN) agonists and antagonists (unspecific: ICI 182 780; specific for ERalpha: methyl-piperidino-pyrazole, MPP). PGI2 and TXA2 production was measured by ELISA. Expression of phospholipases, cyclooxygenases (COX-1 and COX-2), PGI2 synthase (PGIS), and thromboxane synthase (TXAS) was analyzed by western blot and quantitative RT-PCR. E(2) (1-100 nM) dose dependently increased PGI2 production (up to 50%), without affecting TXA2 production. COX-1 and PGIS protein and gene expressions were increased, whereas COX-2, phospholipases, and TXAS expression remained unaltered. All these effects were mediated through ERalpha, since they were produced not only in the presence of E(2), but also in that of PPT, while they were abolished in the presence of MPP. In conclusion, E(2), acting through ERalpha, up-regulates COX-1 and PGIS expression, thus directing prostanoid balance toward increased PGI2 production.

Estradiol stimulates vasodilatory and metabolic pathways in cultured human endothelial cells.

Vascular effects of estradiol are being investigated because there are controversies among clinical and experimental studies. DNA microarrays were used to investigate global gene expression patterns in cultured human umbilical vein endothelial cells (HUVEC) exposed to 1 nmol/L estradiol for 24 hours. When compared to control, 187 genes were identified as differentially expressed with 1.9-fold change threshold. Supervised principal component analysis and hierarchical cluster analysis revealed the differences between control and estradiol-treated samples. Physiological concentrations of estradiol are sufficient to elicit significant changes in HUVEC gene expression. Notch signaling, actin cytoskeleton signaling, pentose phosphate pathway, axonal guidance signaling and integrin signaling were the top-five canonical pathways significantly regulated by estrogen. A total of 26 regulatory networks were identified as estrogen responsive. Microarray data were confirmed by quantitative RT-PCR in cardiovascular meaning genes; cyclooxigenase (COX)1, dimethylarginine dimethylaminohydrolase (DDAH)2, phospholipase A2 group IV (PLA2G4) B, and 7-dehydrocholesterol reductase were up-regulated by estradiol in a dose-dependent and estrogen receptor-dependent way, whereas COX2, DDAH1 and PLA2G4A remained unaltered. Moreover, estradiol-induced COX1 gene expression resulted in increased COX1 protein content and enhanced prostacyclin production. DDAH2 protein content was also increased, which in turn decreased asymmetric dimethylarginine concentration and increased NO release. All stimulated effects of estradiol on gene and protein expression were estrogen receptor-dependent, since were abolished in the presence of the estrogen receptor antagonist ICI 182780. This study identifies new vascular mechanisms of action by which estradiol may contribute to a wide range of biological processes.

Estradiol induces endothelial cell migration and proliferation through estrogen receptor-enhanced RhoA/ROCK pathway.

Migration and proliferation of endothelial cells are involved in re-endothelialization and angiogenesis, two important cardiovascular processes that are increased in response to estrogens. RhoA, a small GTPase which controls multiple cellular processes, is involved in the control of cell migration and proliferation. Our aim was to study the role of RhoA on estradiol-induced migration and proliferation and its dependence on estrogen receptors activity. Human umbilical vein endothelial cells were stimulated with estradiol, in the presence or absence of ICI 182780 (estrogen receptors antagonist) and Y-27632 (Rho kinase inhibitor). Estradiol increased Rho GEF-1 gene expression and RhoA (gene and protein expression and activity) in an estrogen receptor-dependent manner. Cell migration, stress fiber formation and cell proliferation were increased in response to estradiol and were also dependent on the estrogen receptors and RhoA activation. Estradiol decreased p27 levels, and significantly raised the expression of cyclins and CDK. These effects were counteracted by the use of either ICI 182780 or Y-27632. In conclusion, estradiol enhances the RhoA/ROCK pathway and increases cell cycle-related protein expression by acting through estrogen receptors. This results in an enhanced migration and proliferation of endothelial cells.

Cyclooxygenases regulation by estradiol on endothelium.

Estrogen and hormone replacement therapies are being tested to prevent the incidence of cardiovascular disease in postmenopausal women. In spite of the evidence from several epidemiological studies suggesting that estrogens protect against atherosclerosis and associated diseases, controversy exists. Moreover, it is important to develop synthetic compounds that achieve the beneficial effects of estrogens on the cardiovascular system while minimizing such undesirable effects on other tissues as the increased risk of endometrial and breast cancer. Some drugs that modulate estrogen function in a tissue-specific manner (Selective Estrogen Receptor Modulators; SERMs) have been discovered and are currently being used in clinical practice. An example of these is raloxifene. Clinical and experimental data support the consideration of endothelium as a target for estradiol and other sexual hormones. Among other actions, estradiol has been implicated in the control of prostacyclin production through cyclooxygenases (COX) regulation in endothelial cells. Prostacyclins are powerful vasodilators and potent inhibitors of platelet aggregation which are produced from free arachidonic acid through the catalytic activity of two COX: COX-1 and COX-2. Together, these COX represent the main control mechanism for prostacyclin production. Although several non-specific COX inhibitors have been available for decades (aspirin, indomethacin, ibuprofen), COX-2 selective inhibitors have been commercialized only within the last few years, thus making it possible to increase the study and treatment of different disorders. This review will discuss clinical and experimental data that document the endothelial effects of estradiol and SERMs on prostacyclin production and COX regulation, their vascular consequences, and their possible interactions with COX inhibitors.

Raloxifene increases the capacity of serum to promote prostacyclin release in human endothelial cells: implication of COX-1 and COX-2.

Objective:Prostacyclin is a potent vasodilator synthesized by two isoforms of cyclooxygenase in endothelium. The aim of this study was to investigate the effect of serum from postmenopausal women treated with raloxifene on prostacyclin production by human umbilical vein endothelial cells and on cyclooxygenases-1 and -2. Design:Serum was collected from 21 women receiving 60 mg/day of raloxifene, at baseline and at 3 and 6 months. Human umbilical vein endothelial cells were exposed to serum for 24 hours, and prostacyclin production was evaluated in supernatants. Selective inhibitors of cyclooxygenases-1 and -2 (SC-560 and NS-398) were used to investigate the relative contribution of each enzyme. Protein expression for each enzyme was determined using Western blot assays. Results:Prostacyclin production was increased by 30% (P ≤ 0.001) when serum from women treated for 3 and 6 months was added. SC-560 reversed prostacyclin production but did not change baseline values. NS-398, in turn, reduced prostacyclin production using sera from baseline, 3 and 6 months. Cyclooxygenases-1 and -2 protein expression remained unaltered at each treatment step. Conclusions:Serum from women treated with raloxifene stimulated prostacyclin release from human umbilical vein endothelial cells in culture, an effect that seems mediated by increased cyclooxygenase-1 activity.

Selective estrogen receptor modulators and risk for coronary heart disease

Coronary heart disease (CHD) is the leading cause of death in women in most countries. Atherosclerosis is the main biological process determining CHD. Clinical data support the notion that CHD is sensitive to estrogens, but debate exists concerning the effects of the hormone on atherosclerosis and its complications. Selective estrogen receptor modulators (SERMs) are compounds capable of binding the estrogen receptor to induce a functional profile distinct from estrogens. The possibility that SERMs may shift the estrogenic balance on cardiovascular risk towards a more beneficial profile has generated interest in recent years. There is considerable information on the effects of SERMs on distinct areas that are crucial in atherogenesis. The complexity derived from the diversity of variables affecting their mechanism of action plus the differences between compounds make it difficult to delineate one uniform trend for SERMs. The present picture, nonetheless, is one where SERMs seem less powerful than estrogens in atherosclerosis protection, but more gentle with advanced forms of the disease. The recent publication of the Raloxifene Use for The Heart (RUTH) study has confirmed a neutral effect for raloxifene. Prothrombotic states may favor occlusive thrombi at sites occupied by atheromatous plaques. Platelet activation has received attention as an important determinant of arterial thrombogenesis. Although still sparse, available evidence globally suggests neutral or beneficial effects for SERMs.

The risk for cardiovascular disease in women: from estrogens to selective estrogen receptor modulators.

Cardiovascular disease, a generic denomination including coronary heart disease (CHD), stroke, and venous thromboembolic disease (VTED), has shown sensitivity to estrogens. The relative protection of women as compared with men has nourished a debate about a possible protective role for estrogens, but the prejudicial effects detected in clinical trials has created confusion on the risk/benefit ratio induced by hormone administration. The hypothesis that agonists distinct to estrogens might improve the effects associated with estrogens is at the base of the increasing interest on the role of selective estrogen receptor modulators (SERMs). There is a lack of definitive clearcut clinical data on the effects of SERMs in CVD, although the available information suggests a neutral balance on CHD and stroke and an increase in risk similar to estrogens for VTED. Research on pathogenetic mechanisms concentrates in atherosclerosis as the main determinant of the arterial forms of the disease and in hypercoagulability as the counterpart for venous disease. The different experimental models used up to the present moment suggest that, compared with estrogens, SERMs play a less active protection against atherogenesis but do not increase vulnerability of unstable plaques. There is not a clear notion on the mechanisms promoted by SERMs to increase risk for VTED.

Transdermal estradiol reduces F2α-isoprostane levels in postmenopausal women

Objective:F2α-isoprostanes are considered the most reliable index of in vivo oxidative stress. Given the implication of oxidative stress in the pathogenesis of atherosclerosis, we investigated the effects of hormone therapy on the plasma levels of F2α-isoprostanes. Design:Sixty-one healthy postmenopausal women were treated in a randomized trial with estradiol either orally (2 mg/day, 28 women) or transdermally (50 μg/day, 33 women) for 4 weeks. Then women in each group were randomly assigned to oral progestogen, either micronized progesterone (300 mg/day) or medroxyprogesterone acetate (5 mg/day) for 2 additional weeks. Plasma samples were collected before and at the end of each treatment period, either estradiol alone or estradiol plus progestogen. F2α-isoprostanes were measured by a commercial enzyme immunoassay. Results:A significant reduction in the levels of F2α-isoprostanes was detected only in women receiving transdermal estradiol, alone or in combination with medroxyprogesterone acetate. Conclusions:Transdermal estradiol alone or associated with medroxyprogesterone acetate decreased plasma levels of F2α-isoprostanes. These data elucidate additional details of the beneficial effect of estradiol on oxidative stress, a relevant mechanism in atherogenesis.

Progestogens reduce thromboxane production by cultured human endothelial cells

Objectives Progestogens have been poorly studied concerning their roles in endothelial physiology. Prostanoids are vasoactive compounds, such as thromboxane A2, a potent vasoconstrictor, and prostacyclin, a vasodilator. We examined the effects of two progestogens used clinically, progesterone and medroxyprogesterone acetate, on thromboxane A2 production by cultured human umbilical vein endothelial cells (HUVEC) and investigated the role of progesterone receptors and the enzymes involved in production of thromboxane A2 and prostacyclin. Methods Cells were exposed to 1–100 nmol/l of either progesterone or medroxyprogesterone acetate, and thromboxane A2 production was measured in culture medium by enzyme immunoassay. Gene expression of prostacyclin synthase and thromboxane synthase was analyzed by quantitative real-time polymerase chain reaction. Expression of prostacyclin synthase protein was analyzed by Western blot. Results Both progestogens decreased thromboxane A2 release after 24 h. Protein and gene expression of prostacyclin synthase were increased after exposure to both progestogens, without changes in thromboxane synthase expression. These effects induced by progestogens were mediated through progesterone receptors, since they were decreased in the presence of the progesterone receptor antagonist RU486. The cyclo-oxygenase-1 selective inhibitor reduced thromboxane release. Conclusion Progesterone and medroxyprogesterone acetate decreased HUVEC thromboxane release in a progesterone receptor-dependent manner, without changes in thromboxane synthase expression and enhanced prostacyclin synthase gene and protein expression.

Therapeutic dosages of oral or transdermal estradiol did not modify sCD40L levels in postmenopausal women.

The CD40/CD40L system is considered a crucial modulator of the inflammatory process underlying the progression and complication of atheroma plaques. The soluble fraction of CD40L (sCD40L) is a reliable indicator of the CD40/CD40L system. Our purpose was to investigate whether a therapeutic dose of estradiol, by either the oral or the transdermal route, was associated with changes in circulating levels of sCD40L. Forty-seven women completed a 4-week course of treatment with either oral estradiol valerate (2 mg/day, 20 women) or transdermal estradiol (50 μg/day, 27 women). Serum levels of sCD40L were measured by conventional enzyme-linked immunosorbent assay. Oral, but not transdermal estradiol, modified the lipid profile. Levels of sCD40L, however, remained unchanged compared with baseline. This neutral effect of oral or transdermal estradiol on sCD40L levels further advances our knowledge on the effects of estrogens on mechanisms involved in the progression and complication of atherosclerosis.